Bioactivity of a peptide derived from acetylcholinesterase: involvement of an ivermectin-sensitive site on the alpha 7 nicotinic receptor

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Abstract

A peptide fragment of 14 amino acids, derived from the C-terminus of acetylcholinesterase (AChE), might underlie the now well-established noncholinergic effects of the enzyme. This peptide is bioactive in a variety of systems including acute (brain slices) and chronic (organotypic culture) preparations of hippocampus, a pivotal area in Alzheimer's disease (AD); invariably, the action of the peptide is mediated specifically via an as yet unknown receptor. In this study, the allosteric alpha 7 agent, ivermectin (IVM), had a modest inhibitory effect, whilst that of the peptide was significantly more marked. However, ivermectin rendered ineffective the toxicity of high doses of the peptide, that is, when the two were co-applied, only the smaller effects of ivermectin were seen. Ivermectin, therefore, is presumably acting at a site that is identical to, or at least strongly interactive with, the normal binding site for AChE-peptide. This observation could have important implications for eventual therapeutic targeting of the action of AChE-peptide, in neurodegeneration.

Introduction

It is well know that acetylcholinesterase (AChE) has noncholinergic functions in relation to development Appleyard, 1992, Arendt et al., 1992, Day and Greenfield, 2002, Greenfield, 1991, Greenfield, 1996, Soreq and Seidman, 2001 possibly by enhanced calcium influx (Webb et al., 1996). The parallels between AChE, acting in a nonclassical manner, and actions of APP/β-amyloid Appleyard et al., 1987, Farlow et al., 1992, Furukawa et al., 1996, Greenfield, 1995, Jones et al., 1995, Milward et al., 1992, Webb et al., 1996, Wu et al., 1995 have led to the identification of a domain, AEFHRWSSYMVHWK (AChE-peptide), located toward the “C” terminus of AChE, which has a strong homology with APP (Greenfield and Vaux, 2002).

A candidate receptor for the functional effects of noncholinergic AChE is the α7 nicotinic receptor. Indeed, this highly calcium-permeant receptor (Seguela et al., 1993) is transiently expressed in rat brain in parallel with AChE (Broide et al., 1996) and, moreover, strongly binds proteins Wang et al., 2000a, Wang et al., 2000b closely related to AChE (Greenfield and Vaux, 2002).

To date, most is known about allosteric modulation on the α4β2 subunit; however, there are also some recent reports of modulation of the α7 nicotinic acetylcholine receptor (nAChR). For example, ivermectin (IVM) acts as an allosteric effector that binds to a specific site on the α7 nAChR: micromolar concentrations of IVM markedly enhance ACh-evoked currents of the neuronal chick and human α7 nAChR in oocytes (Krause et al., 1998). Hence, we have first confirmed that in the hippocampus, IVM does indeed act primarily at alpha-7 receptors, and we have then tested whether the bioactive effects of AChE-peptide could be modified by IVM.

The preparation chosen as most appropriate was one where not only the peptide is effective, but where the noncholinergic actions of AChE have been well-documented: mammalian organotypic cultures Day and Greenfield, 2002, Holmes et al., 1997, Jones et al., 1995. Since noncholinergic acetylcholinesterase is well known to affect the hippocampus Appleyard, 1992, Day and Greenfield, 2002, Small et al., 1996 and since this region is so important in Alzheimer's disease (AD), the effects of AChE-peptide on neurite outgrowth were investigated at different concentrations and for different periods of time. These actions were compared with those of “scrambled” peptide sequence HSWRAEVFHKYWSM to control nonspecific bulk peptide effects.

Section snippets

Materials and methods

Materials were obtained as follows: Hank's balanced salt solution, Eagle's MEM, horse serum, poly-d-lysine, chicken plasma, bovine thrombin, glutamine, and mitotic agents were obtained from Sigma-Aldrich Company Ltd (Poole, UK). Fungizone was obtained from Life Technologies Ltd (Paisley) and ivermectin from Tocris Cookson Ltd (UK). All peptides used were synthesized at the Dyson Perrins laboratory for organic chemistry (Dr. M. Pitkeathley, University of Oxford) using an Applied Biosystems 430A

Characterization of ivermectin: 125I-αBTX binding

To prove the selectivity of IVM for α7 nAChR, we tested the effects of IVM on 125I-αBTX binding on rat hippocampal membranes. The presence of binding sites for 125I-αBTX, a specific ligand of muscle and α7 nAChR subtypes (Lindstrom, 2000), would be consistent with the expression of the α7 nAChR subtypes. As shown in Fig. 1, IVM inhibited 125I-αBTX binding at relatively low micromolar concentrations.

Selectivity of AChE-peptide

The scrambled variant of the peptide had no effect on neurite outgrowth in the hippocampal

Procedural considerations

The organotypic culture system was chosen as a model that resembles the in vivo environment as closely as possible, whilst offering the accessibility and reductionism of the in vitro preparation. Some of the advantages of this system include maintenance in a high degree of organotypic organization, cellular differentiation, and tissue-specific cell connections Gahwiler, 1981, Gahwiler, 1984, Caeser and Aertsen, 1991, Zimmer and Gahwiler, 1984, as well as preservation of local neuronal circuits

Acknowledgements

This work was supported by Synaptica Limited and University of Oxford.

We would like to thank Drs. Toni Day and Steve Butcher for their helpful advice on the techniques.

References (55)

  • Z.Z. Guan et al.

    Selective changes in the levels of nicotinic acetylcholine receptor protein and of corresponding mRNA in the brains with Parkinson's disease

    Brain Res.

    (2002)
  • V.D. Guerguiev et al.

    Involvement of α7 nicotinic acetylcholine receptors in activation of tyrosine hydroxylase and dopamine β-hydroxylase gene expression in PC12 cells

    J. Neurochem.

    (2000)
  • F.J. Munoz et al.

    Neurotoxicity of acetylcholinesterase amyloid beta-peptide aggregates is dependent on the type of Abeta peptide and the AChE concentration present in the complexes

    FEBS Lett.

    (1999)
  • F.J. Munoz et al.

    Peripheral binding site is involved in the neuropathic activity of acetylcholinesterase

    Neuroreport

    (1999)
  • R.L. Papke et al.

    α7 Receptor-selective agonists and modes of α7 receptor activation

    Eur. J. Pharmacol.

    (2000)
  • A. Robitzki et al.

    Regulation of cholinesterase gene expression affects neuronal differentiation as revealed by transfection studies on reaggregating embryonic chicken retinal cells

    Eur. J. Neurosci.

    (1997)
  • P. Seguela et al.

    Molecular cloning, functional-properties, and distribution of rat brain α7 receptor—A nicotinic cation channel highly permeable to calcium

    J. Neurosci.

    (1993)
  • K.V. Sharma et al.

    Direct evidence for an adhesive function in the noncholinergic role of acetylcholinesterase in neurite outgrowth

    J. Neurosci. Res.

    (2001)
  • A.D. Smith et al.

    Alzheimer's disease and acetylcholinesterase-containing neurons

    Lancet

    (1984)
  • H. Soreq et al.

    Acetylcholinesterase—New roles for an old actor

    Nat. Rev., Neurosci.

    (2001)
  • H. Wang et al.

    β-Amyloid1–42 binds to α7 nicotinic acetylcholine receptor with high affinity

    J. Biol. Chem.

    (2000)
  • H. Wang et al.

    Amyloid peptide Aβ-1–42 binds selectively and with picomolar affinity to α7 nicotinic acetylcholine receptors

    J. Neurochem.

    (2000)
  • J. Wu et al.

    Beta-amyloid selectively augments NMDA receptor-mediated synaptic transmission in rat hippocampus

    NeuroReport

    (1995)
  • M.E. Appleyard et al.

    Cholinesterase activities in cerebrospinal fluid of patients with senile dementia of Alzheimer type

    Brain

    (1987)
  • J.W. Bigbee et al.

    Morphogenic role for acetylcholinesterase in axonal outgrowth during neural development

    Environ. Health Perspect.

    (1999)
  • S. Brimijoin et al.

    Cholinesterases in neural development: new findings and toxicologic implications

    Environ. Health Perspect

    (1999)
  • R.S. Broide et al.

    Regulation of α7 nicotinic acetylcholine receptors in the developing rat somatosensory cortex by thalamocortical afferents

    J. Neurosci.

    (1996)
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